FCS/SIFT measurements

For this assay the expression and purification of α-Syn and sumoylated α-Syn was performed as described previously (Krumova et al. 2011). The labelling of both proteins with Alexa Fluor-647-O-succinimidylester (Molecular Probes®, USA) was carried out as described previously (Giese et al. 2005). Green labelled small unilamellar Dipalmitoyl-sn-glycero-3-phospho-choline lipid vesicles (DPPC-SUV) were generated as described previously (Högen et al. 2012). Scanning for intensely fluorescent targets (SIFT) and Fluorescence correlation spectroscopy (FCS) measurements for the quantification of α-Syn vesicle binding were performed with an Insight Reader (Evotec-Technologies) with dual colour excitation at 488 and 633 nm as described before (Högen et al. 2012). All measurements were carried out after an incubation period of at least 30 min of DPPC-SUV with labelled α-Syn. For equilibrium conditions, measurements were performed at least 2 h after addition of unlabelled non-sumoylated α-Syn.

Materials and Methods 43

2.2.4.4. Electrochemiluminescence assay for quantification of α-Synuclein

For the quantification of α-Syn in cell lysates and EVs, derived from primary neurons, a slightly modified electrochemiluminescence assay was used (Kruse et al. 2012). Briefly, the antibody MJF-1, clone 12.1 (kindly provided by Dr. Liyu Wu, Epitopics Burlingame, USA), was coated on standard 96-well Multi-Array plates (Meso Scale Discovery, Gaithersburg, USA) and incubated over night at 4°C. All additional steps were performed at room temperature. The plates were washed three times with 150 µL PBS + 0.05 % Tween-20.

Subsequent blocking was performed with 150 µg BSA (Meso Scale Discovery, Gaithersburg, USA) for 1 h with gently shaking at 300 rpm. A serial four-fold dilution of recombinant α-Syn (kindly provided by Dr. Omar el- Agnaf, United Arab Emirates University, Al Ain, United Arab Emirates), starting at 25.000 pg/ml, was used to prepare a standard curve. After washing as indicated above, 25 µL of standards and samples were applied per well in duplicates. To secure a successful binding of the antibody to the samples, the plates were shaking for 1 h at 700 rpm and then washed again as indicated above. Afterwards addition of 25 µL of Sulfo-TAG labelled anti α-Syn clone 42 (BD Transduction Laboratories, Heidelberg, Germany) was added to achieve a final concentration of 1 µg/mL and incubated for 1 h at 700 rpm. Three washing steps followed, before 150 µL of 2 x Read Buffer (Meso Scale Discovery, Gaithersburg, USA) was applied to each well and the plates were measured in a Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, USA). The final data analysis was performed using MSD Discovery Workbench 3.0 Analysis Toolbox.

2.2.4.5. Labelling of SUMO-2 with the ESPIT dye MFM

SUMO-2 was labelled at its single cysteine (Cys) 52 with the ESPIT (excited state intramolecular proton transfer) probe MFM (Shvadchak et al. 2011). To uncover the Cys 52, SUMO-2 was pre-treated with 1 mM DTT and a buffer exchange to 25mM PO₄-Na, pH 6.5, without any sulfhydryl groups. Afterwards the protein concentration was measured and adjusted between 200 µM and 350 µM, followed by the addition of the MFM dye (1-4 mg/mL) in 1-2 times excess and an incubation period for 12-24 h with gently mixing at 4°C. Finally, 10 times excess of N-Methylmaleimide in DMSO was added and incubated for 30min in the same conditions as before. This step is necessary to block any remaining free Cys groups.

For purification the labelled protein was applied to a gravity PD 10 column (GE Healthcare Ltd., Little Chalfont, Buckinghmanshire, UK) and eluted with the same buffer, while collecting fractions of 5-10 drops. The fractions were checked for absorbance with a NanoDrop (PEQLAB Biotechnologie GmbH, Erlangen, Germany), pooled into one tube, aliquoted in small volumes and flash frozen in liquid N₂. The labelled protein was stored at -20°C.

Materials and Methods 44

2.2.5. Lipid biochemistry

2.2.5.1. Preparation of Small Unilamellar Vesicles (SUVs)

Small Unilamellar Vesicles (SUVs) were prepared by sonication as described previously (Huang et al. 1974), (Storch et al. 1986), (Falomir-Lockhart et al. 2011). Briefly, the composition of SUVs based on mixtures of POPC, POPC and PIPS (Avanti Polar Lipids, Inc., Alabaster, AL, USA). The relative molar compositions and approximate charge densities were as follows (POPC, 100; [0], POPC:POPS, 90:10; [-0.1], POPC:POPS:PI(3)P 85:10:5;

[-0.13], POPC:POPS:PI(5)P 85:10:5; [-0.14); POPC:POPS:PI(3,5)P2 85:10:5; [-0.2];

POPC:POPS:PI(4,5)P₂ 85:10:5, [-0.2] and POPC:POPS:PI(3,4,5)P₃, 85:10:5, [-0.25]).

At first the lipids were mixed from their chloroform stocks, in molar ratios indicated above, in clean glass balloons, followed by drying the mixture under a gently stream of nitrogen.

Afterwards the dry lipid mixture was resuspended in a specific volume of buffer (25 mM HEPES, 100 mM KCl, pH 7.26) and transferred to a falcon tube and sonicate in an ice water bath at least for 30 min, until the solution appeared translucent.

After a 1 h centrifugation at 4°C and maximum speed, the vesicles were stored at least 5°C above the transition temperature of the lipid mixture and used within 10 days of their preparation. The vesicles were quantified by determining the inorganic phosphorus (Gomori 1942).

2.2.5.2. Membrane binding assay of SUMO-2

The measurements of labelled SUMO-2-MFM with SUVs were performed with a new 96 well microplate slope assay (to be published elsewhere). This assay offers several advantages compared to conventional fluorescence assays as lipids are added to proteins. Thereby, e.g.

emission and scattering from lipids, photo-bleaching effects during the sequential addition of lipids and waste of material are avoided.

The strategy of “slopes” takes advantage of the maximal sensitivity of a titration performed with lipid concentrations in excess varied around the anticipated value of the dissociation constant K_dS. The slopes measured for a small number of protein concentrations are plotted versus the lipid concentrations, from which K_d and the fluorescence enhancement factor are calculated from the relation: slope = f0[1+(fe-1) α, where f0 is the slope corresponding to 0 lipid concentration and fe is the (enhanced) fluorescence of the bound protein relative to that of the free protein. Some major advantages of this assay are: the parallel readout in a microplate reader, the possible bottom readout with a small optical path length and therefore minimal scattering effects. Additionally, it is enough to use a minimal amount of reagents, endpoint determinations, that means no photo- bleaching effects.

Materials and Methods 45

Solutions of SUMO-2-MFM (100 nM, 200 nM and 300 nM) were prepared with 7 different SUV concentrations (0-120 µM) in 25 mM HEPES, 100 mM KCl, pH 7.26. Afterwards 100 µl of these 48 mixtures were added in duplicates to a 96 well quartz microplate (Hellma Analytics, Müllheim, Germany). After an incubation period of at least 10 min at room temperature, the fluorescence was recorded at 540 nm in BMG Pherastar plate reader (BMG Labtech, Ortenberg, Germany). The recording was applied with a bottom readout, well scan mode with a 10 x 10 matrix, a well scan diameter of 5 mm and with 25 flashes per well. Wells without lipid and/or protein were included to the data sets in order to establish blank values and the lipid contributions to the measured signal. Finally the data were analyzed with procedures implemented in Mathematica (Wolfram Research).

2.2.6. NMR spectroscopy

In order to study membrane binding of SUMO-2 NMR spectroscopy was performed. Thus 200 µM of ¹⁵N-labelled SUMO-2 in 20 mM NaH₂PO₄/Na₂HPO₄, pH 6.8, 100 mM KCl, 1 mM DTT was titrated with increasing concentrations (8, 16 and 32 mM) of DHPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine). ¹H, ¹⁵N-HSQC spectra were acquired at 600 MHz and 22 ºC on a triple resonance room temperature probe with 16 transients, 2084 x 256 total points and widths of 8418 x 2129 Hz (¹H x ¹⁵N). Carrier frequencies were set to the water resonance for

1H and to 117 ppm for ¹⁵N. Resonance assignments were taken from BMRB entry 11267.

The normalized weighted average chemical shift difference for the amide proton and nitrogen were calculated accordingto



(HN)

= [

H²

+

(0.2

*

N

)

²

]

^1/2

.

2.2.7. Immunocytochemistry

2.2.7.1. Immunofluorescence staining

Proteins were labeled with specific primary antibodies and fluorophore-labeled secondary antibodies to determine their localization in cultured N2a cells. All steps of the staining protocol were carried out at RT. N2a cells were grown on PLL-coated glass coverslips, washed once with PBS and fixed then with PFA (Paraformaldehyde) (4 % PFA in PBS, pH 7.4) for 25 min. Thereafter coverslips were washed three times with PBS and cells were permeabilized in 0.1 % (v/v) Triton X-100 (in PBS), that allows the antibodies to enter the cell. Subsequently the cells were washed immediately three times and covered with 100 % blocking solution (see below) for 35 min to avoid unspecific binding of the antibodies.

Materials and Methods 46

Primary antibodies (see Table 2) were diluted in 10 % blocking solution and incubated with the cells in a dark and humidity chamber for 1 h at RT. After three washing steps with 1 x PBS for 5 min, cells were incubated with fluorophor-conjugated secondary antibodies in 10 % blocking solution for 1 h, again in a dark and humidity chamber. Thereafter, the cells were washed 3 times with 1 x PBS for 5 min and once with bi- distilled H2O to remove remaining salt traces, followed by mounting the glass coverslips onto glass slides with a drop of mowiol (see below) and dried overnight. For long term period the slides were kept in the dark and stored at 4°C.

100 % Blocking solution 2 % BSA

2 % FCS

0.2 % Gelatin, from cold water fish skin add 10 mL 10 x PBS

Fill up to 100 mL with bi- distilled H2O.The solution was aliquoted to 5 mL and stored at -20°C.

Preparation of 16 % paraformaldehyde (PFA)

For the preparation of 16 % paraformaldehyse (PFA) solution, 16 g PFA (AppliChem GmbH, Darstadt, Germany) was mixed with 70 mL bi-distilled H₂O and dissolved by heating to 60°C.

Thereafter 2-3 pellets NaOH were added, resulting in a noticeable cooling of the solution, followed by the addition of 10 mL 10 x PBS and the chilling to room temperature. Finally the pH was adjusted to 7.4 and the solution was filled up to 100 mL with bi-distilled H₂O. The solution was separated to 3 mL aliquots and stored at -20°C.

Preparation of mowiol solution

To prepare the mounting solution, 2.4 g mowiol (GmbH, Darstadt, Germany) and 6 g glycerol were mixed and incubated at room temperature for 2 h with gentle agitation. Thereafter, 12 mL 0.2 M Tris/HCl (pH 8.5) were added and the solution was mixed under heating to 50°C. A subsequent centrifugation step at 5,000 x g secure the clearance of the solution, followed by the addition of the anti-fading reagent 1,4-Diazabicyclo[2.2.2] octan (DABCO) in a final concentration of 24 mg/ml (Sigma-Aldrich St. Louis, MO, USA). Finally the mowiol solution was aliquoted and stored at -20°C.

Materials and Methods 47

2.2.8. Microscopy

2.2.8.1. Confocal microscopy

To visualize and record the localization of proteins, which were stained with fluorescent antibodies, in PFA fixed cells, confocal microscopy was applied. The images were acquired with a Leica DMIRE2 microscope with a 63 x oil-immersion objective and a Leica TCS SP2 AOBS confocal laser scanning setup (Leica Microsystems, Darmstadt, Germany).

2.2.8.2. Electron microscopy

EVs were prepared from cerebrospinal fluid and culture medium as described in section 2.2.3.1. The 100,000 x g pellet was fixed with 4% PFA and was adsorbed to glow-discharged Formvar-carbon-coated copper grids by floating the grid for 10 min on 5 µl droplets on Parafilm. The grids were negatively stained with 2% uranyl acetate containing 0.7 M oxalate, pH7.0, and imaged with a LEO EM912 Omega electron microscope (Carl Zeiss, Jena, Germany). Digital micrographs were obtained with an on-axis 2048 x 2084 CCD camera (Proscan GmbH, Scheuring, Germany). (Electron microscopic imaging of EVs was kindly performed by Dr. Wiebke Möbius, MPI for experimental medicine, Göttingen).

2.2.9. Image processing and statistical analysis

2.2.9.1. Quantification of extracellular vesicle secretion

To compare the relative EV release, EV pellets and the corresponding cell lysates were subjected to Western blotting as described in section 2.2.4.1 and 2.2.4.2. After developing of the Western blot membranes on X-ray films (CL-XPosure™ Film, Thermo Fisher Scientific, Rockford,IL, USA), the films were scanned and analysed with ImageJ software for the signal intension of protein bands on the X-ray films. As a degree of EV release, the ratio of signal intensities of EVs versus corresponding cell lysates was calculated from at least 4-13 independent experiments.

Materials and Methods 48

2.2.9.2. Statistical analysis

Data were statistical analysed with MS Office Excel 2007 (Microsoft Deutschland GmbH, Berlin, Germany). For descriptive statistics, mean and standard error of the mean (SEM) of a data set were calculated and illustrated with MS Office Excel 2007. For the comparison of two independent groups with normal distribution of sample sets and equal variance, the parametric Student's t-test was used. A data group which displays a p-value less than 0.05 was regarded as significantly different.

Results 49

3. Results

Most of these results have been published in:

Extracellular vesicle sorting of α‐Synuclein is regulated by sumoylation

Marcel Kunadt, Katrin Eckermann, Anne Stuendl, Jing Gong, Belisa Russo Katrin Strauss, Surya Rai, Sebastian Kügler, Lisandro Falomir Lockhart, Martin Schwalbe, Petranka Krumova, Luis M. A. Oliveira, Mathias Bähr, Wiebke Möbius, Johannes Levin, Armin Giese, Niels Kruse, Brit Mollenhauer, Ruth Geiss-Friedlander, Albert C. Ludolph, Axel Freischmidt, Marisa S. Feiler, Karin M. Danzer, Markus Zweckstetter, Thomas M. Jovin, Mikael Simons, Jochen H. Weishaupt, Anja Schneider

Acta Neuropathol DOI 10.1007/s00401-015-1408-1

The results displayed in Fig. 9 A, Fig. 10 A, Fig. 11, Fig. 13, Fig. 17 and Fig. 18 were first performed by Surya Rai, a former master student under the supervision of Prof. Dr. Anja Schneider. In the course of this thesis, the experiments were repeated to increase the number of performed experiments and to improve the significance.

3.1. α-Synuclein is released in extracellular vesicles

In neurodegenerative diseases extracellular vesicles (EVs) have been proposed to be potential carriers of misfolded proteins and thereby may be responsible for the spreading of the disease pathology (Aguzzi et al. 2009). In this study we aimed to investigate how α-Syn is sorted into EVs.

3.1.1. α-Synuclein is released in extracellular vesicles derived from N2a cells

For the preparation of EVs, the conditioned medium was collected and subjected to subsequent centrifugation steps (see section 2.2.2.5 and section 2.2.3.1). In a final ultracentrifugation step at 100.000 x g for 1 h, EVs were pelleted as previously described (Trajkovic et al. 2008). We further refer to this 100.000 x g pellet as EV pellet (P100). The P100 and the cell lysate of the corresponding secreting parental cells were subjected to Western blot analysis and probed with an antibody against α-Synuclein. As shown in Fig. 6 A α-Syn was enriched in the P 100.

Results 50

As a positive control the EV fraction and the corresponding lysates were also stained with the EV marker proteins Alix (AIP-1) and Flotilin 2 (Flot-2). In addition to the signal for α-Syn we also found intense signals for both EV marker proteins, Alix and Flot-2 in the P100. A contamination of the P100 with cellular compartments, membrane particles or other vesicles than EVs could be excluded by the absence of a signal for cellular compartments, like the ER marker protein Calnexin (Fig. 6 A).

Fig. 6: α- Synuclein is released in extracellular vesicles derived from N2a cells

(A) Cultured medium of N2a cells was collected and subjected to subsequent centrifugations steps to clear the medium from cell debris, dead cells and macrovesicles with 1 x 10 min at 3500 x g, 2 x 10 min at 4500 x g and 1 x 30 min at 10,000 x g. In a final centrifugation step the EVs were pelleted. The whole EV pellet and 10 µl of the corresponding cell lysates were subjected to Western Blot analysis. The P100 pellet is immune positive for α-Syn and the EV marker proteins Flot-2 and Alix, but negative for the ER marker Calnexin. (B) For a broader purification the P100 was loaded on top of a sucrose gradient (1.03-1.32 g/mL) and ultracentrifuged for 16 h at 200,000 x g. The collected fractions were ultracentrifuged again and the pellets as well as the corresponding lysates were subjected to Western Blot analysis and immune stained against α-Syn and Alix. The detected signals corresponded to known densities for EVs ranging from 1.11 to 1.20 g/mL. (C) EVs derived from N2a cells were processed to electron microscopy and showed their typical cup shaped morphology (scale bar 100 nm).

In another experiment we subjected the P100 to sucrose density ultracentrifugation, to get a higher purity level of the EV fraction as well as to further characterise the previous P100. The gradient was centrifuged at 200,000 x g for 16 h. After the ultracentrifugation step 8 fractions, corresponding to densities between 1.03-1.32 g/mL (0.25-2.5 M), were collected and diluted 1:6 with PBS. These fractions were processed to Western Blot analysis and immunostained for a-Syn and for the EV marker protein Alix. As shown in Fig. 6 B signals were detected for α-Syn in the fraction of 1.20 g/mL and for Alix in fractions of 1.11, 1.16 and 1.20 g/mL.

Results 51

This is in line with the previous described flotation behaviour of EVs (Fauré et al. 2006, Théry et al. 2006). To visualize EVs we subjected the 100.000 x g pellet to electron microscopy and negatively stained the pellets with 1 % uranyl acetate. We found the typical cup shaped morphology (Simons et al. 2009) with diameter between 50 nm and 100 nm, as previously observed by transmission and cryo-electron microscopy (Conde-Vancells et al. 2008) (Fig.

6 C). Taken together, these data demonstrate that α-Syn is released within EVs derived from N2a cells and that we are able to recover material with our EV purification protocol.

3.1.2. α-Synuclein is localized in extracellular vesicles in vivo

It is not known whether α-Syn is present in EVs in vivo. To address this issue we firstly analysed whether α-Syn is present in EVs in the human central nervous system (CNS).

Therefore, we prepared EVs from cerebrospinal fluid (CSF) after the written informed consent was given of patients with PD. Analysis of patient CSF was approved by the ethical committee of the Medical Faculty, University Medicine Goettingen (IRB 02/05/09). The CSF was subjected to a series of centrifugation steps to clear the CSF from cell debris with 1 x at 3500 x g for 10 min (P3), 2 x at 4500 x g for 10 min (P4), 1 x at 10.000 x g for 30 min (P10) and a final 100.000 x g ultracentrifugation step (P100). Pellets of each centrifugation step and the EV pellet (P100) were subjected to Western blot analysis and probed with Flot-2 and Calnexin antibodies. As shown in Fig. 7 A Flotillin 2 was enriched in the EV fraction and a contamination of the 100,000 x g pellet could be excluded by immunostaining for the ER marker Calnexin.

Results 52

Fig. 7: Characterization of extracellular vesicles in cerebrospinal fluid

(A) Cerebrospinal fluid was processed to a series of centrifugation steps and each fraction as well as the P 100 was immunostained in Western blot. (B) Part of the P 100 was negatively stained with 1 % uranyl acetate and the EVs were visualized by electron microscopy (scale bar 100 nm). (C) Immunostaining of 100.000 x g pellets against various microsomal and EV marker proteins. (D) Discontinuous sucrose density gradient (0.25 M-2.5 M) was analysed by Western Blot for the presence of Flot-2. (E) EVs were prepared from 5 mL CSF and 20 µL of total CSF and the corresponding 100.000 x g pellet were subjected to Western blot analysis. One representative blot out of 3 different patient samples is shown. (F) A 100,000 x g pellet of a Parkinson dementia CSF sample was loaded on a discontinuous sucrose gradient (0.25 M-2.5 M) and α-synuclein was quantified in each fraction via an electrochemiluminescence assay.

Electron microscopy of the resulting P100 revealed 50-100 nm structures with the typical cup shaped morphology for EVs (Fig. 7 B). The 100.000 x g pellet was also enriched for EV marker protein CD63 as well as for the Glutamate Receptors- 1, -2 and -3. This latter indicates that CSF EVs are at least partially derived from the central nervous system.

Microsomal proteins such as the ER marker Calnexin and the trans golgi network (TGN) protein -Adaptin were absent (data not shown), thus excluding microsomal contamination of the EV preparation (Fig. 7 C). On a sucrose gradient Flotillin-2 positive EVs showed a consistent floating behaviour as supported by previously published results (Baietti et al.

2012). Flotillin-2 was enriched at a density of 1.16-1.24 g/mL (Fig. 7 D).

Results 53

To elucidate whether α-Syn is enriched in the P100 of CSF in comparison to total cerebrospinal fluid, we processed total CSF and the corresponding 100.000 x g pellet to Western blot analysis and the samples were immunostained for α-Syn. As shown in Fig. 7 E, the 100,000 x g pellet revealed an enriched α-Syn signal compared to total CSF. In addition we performed a sucrose density ultracentrifugation experiment with a 100,000 x g pellet of a Parkison’s disease CSF sample. In this experiment EVs of CSF samples were isolated and the resulting 100.000 x g pellet was subjected to a discontinuous sucrose gradient, consisting of 8 different layers (0.25 M-2.5 M, see section 2.1.6.3). Subsequent detection of α-Syn by an electrochemiluminescence assay (see section 2.2.4.4) revealed flotation behaviour of CSF derived α-Syn, similar to the EV marker protein Flotilin-2 (Fig. 7 F). Taken together, all these findings indicate, that α-Syn associated EVs are present in the CNS in vivo

3.1.3. α-Synuclein is predominantly localized in the lumen of EVs

We next wanted to clarify whether α-Syn is either localized in the lumen of EVs or rather attached to the outer membrane. To this end, we transiently transfected N2a cells with a wild-type α-Syn plasmid and EVs were prepared from cultured medium and processed to subsequent centrifugations steps, as described previously in section 2.2.3.1. The P100 was resuspended in PBS and divided into two equal parts. One part was digested with trypsin and the other only with PBS as a control. The silver gel shows degradation bands for the trypsin treated P100 pellet compared to the non-trypsinized control (PBS treated) (Fig. 8 A).

Western Blot analysis showed that the content of the bona fide intraluminal protein Flotilin-2 and α-Syn was unaltered by trypsin treatment, which indicates that α-Syn is localised in the lumen of EVs (Fig. 8 B).

Results 54

Fig. 8: α-Syn is localised in the intraluminal compartment of extracellular vesicles

Fig. 8: α-Syn is localised in the intraluminal compartment of extracellular vesicles

Im Dokument Extracellular vesicle release of α-Synuclein is mediated by SUMOylation (Seite 57-0)